Fig. 6: SEC and SPY promote the SPT-mediated repression of PID transcription rather than its ability to form homo- and heterodimers.
From: O-glycosylation of the transcription factor SPATULA promotes style development in Arabidopsis
a, Left, FRET–FLIM assay showing that both SEC and SPY have no effect on the strength of the interaction between SPT–GFP and its interacting partners SPT–RFP, IND–RFP and HEC1–RFP, in nuclei of co-infiltrated tobacco leaves. The experiments were repeated at least three times for each combination; more than 60 nuclei were used for quantification for each combination. Right, the FRET efficiency (%) is indicated in the violin plots. The internal dashed and dotted lines indicate the median and quartiles. Different letters indicate significant differences (P < 0.05) tested by ordinary one-way ANOVA multiple comparisons. The exact P values are provided in the Source data. b, Top, schematic representation of the 1-kb promoter of PID used in the transactivation assays, including the TF binding sites (G-boxes, indicated by the red lines, and E-box, indicated by the blue line). Middle, quantification of GUS expression by qRT-PCR experiments showing that both SEC–HA and SPY–HA enhance the transcriptional downregulation triggered by SPT and IND. The optical density (OD) values employed were OD = 0.1 for SPT–GFP and IND–RFP and OD = 0.5 for SEC–HA and SPY–HA, in each experiment. The experiments were repeated three times for each combination (n = 3). The values shown are means ± s.d. Different letters indicate significant differences (P < 0.01) tested by ordinary one-way ANOVA multiple comparisons. The exact P values are provided in the Source data. Bottom, immunodetection of SPT–GFP, IND–RFP, SEC–HA and SPY–HA from N. benthamiana Agrobacterium-infiltrated leaves used for the transactivation assays. The Coomassie brilliant blue (CBB) bands were used as a sample loading control. c, EMSA experiments showing that SEC (5TPR–SEC) and SPY (3TPR–SPY) both enhance the binding of SPT (6xHis–SPT) to the region of the PID promoter (171-bp pPID). Similar results were obtained from two independent experiments. d, Schematic working model showing that SEC and SPY act upstream of SPT to attach O-GlcNAc (blue square) and O-fucose (red triangle), which in turn promotes the binding of SPT to the PID promoter and/or its transcriptional activity. In this way, SEC and SPY contribute to the fine-tuning of auxin distribution while repressing CK sensitivity at the gynoecium apex, ultimately promoting style morphogenesis.
